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Byline: Nao Shimada (1), Keiko Nishio (2), Mineko Maeda (2), Hideko Urushihara (3), Takefumi Kawata (1) Keywords: Dictyostelium; Extracellular matrix protein; In situ hybridization; Morphogenesis; Transcription factor Abstract: Dd-STATa is a functional Dictyostelium homologue of metazoan STAT (signal transducers and activators of transcription) proteins, which is activated by cAMP and is thereby translocated into the nuclei of anterior tip cells of the prestalk region of the slug. By using in situ hybridization analyses, we found that the SLF308 cDNA clone, which contains the ecmF gene that encodes a putative extracellular matrix protein and is expressed in the anterior tip cells, was greatly down-regulated in the Dd-STATa-null mutant. Disruption of the ecmF gene, however, resulted in almost no phenotypic change. The absence of any obvious mutant phenotype in the ecmF-null mutant could be due to a redundancy of similar genes. In fact, a search of the Dictyostelium whole genome database demonstrates the existence of an additional 16 homologues, all of which contain a cellulose-binding module. Among these homologues, four genes show Dd-STATa-dependent expression, while the others are Dd-STATa-independent. We discuss the potential role of Dd-STATa in morphogenesis via its effect on the interaction between cellulose and these extracellular matrix family proteins. Author Affiliation: (1) Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan (2) Department of Biology, Graduate School of Science, Osaka University, Osaka, Japan (3) Biological Sciences, University of Tsukuba, Ibaraki, Japan Article History: Registration Date: 24/06/2004 Received Date: 20/05/2004 Accepted Date: 18/06/2004 Online Date: 10/08/2004

During development, a few Dictyostelium cells spontaneously emit pulses of cAMP, and surrounding cells respond within a minute by releasing more of the signal. When observed in monolayers, cAMP relay [...], Self-regulating systems often use robust oscillatory circuits. One such system controls the chemotactic signaling mechanism of Dictyostelium, where pulses of adenosine 3',5'-monophosphate (cAMP) are generated with a periodicity of 7 minutes. We have observed spontaneous oscillations in activation of the mitogenactivated protein (MAP) kinase ERK2 that occur in phase with peaks of cAMP, and we show that ERK2 modulates cAMP levels through the phosphodiesterase RegA. Computer modeling and simulations of the underlying circuit faithfully account for the ability of the cells to spontaneous[y generate periodic pulses during specific stages of development. Similar oscillatory processes may occur in cells of many different species.

Northern blot and sequence analyses of a developmental mutant in Dictyostelium discoideum reveals that cell differentiation and transport of the chemotactic signal of cAMP are affected by a protein kinase of the MAP kinase/ERK family, leading to the disruption of morphogenesis. cGMP synthesis occurs at normal levels while cAMP is synthesized at very low levels by ERK2 encoding locus-disrupted strains in response to a cAMP stimulus. The detected kinase protein participates in the activation of adenylyl cyclase and is essential for normal Dictyostelium development.

We analyzed the spatial expression patterns of the genes involved in myosin function by in situ hybridization at the tipped aggregate and early culmination stages of Dictyostelium. Myosin heavy chain II mRNA was enriched in the anterior prestalk region of the tipped aggregates, whereas it disappeared from there and began to appear in both upper and lower cups of the early culminants. Similarly, mRNAs for essential light chain, regulatory light chain, myosin light chain kinase A, and myosin heavy chain kinase C were enriched in the prestalk region of the tipped aggregates. However, expression of these genes was distinctively regulated in the early culminants. These findings suggest the existence of mechanisms responsible for the expression of particular genes, [C] 2000 Academic Press Key Words: in situ hybridization; myosin heavy chain II; myosin light chain; myosin light chain kinase; myosin heavy chain kinase; upper cup; Dictyostelium.